The cytochromes P-450 metabolize a wide array of compounds, including xenobiotics such as drugs and carcinogens, and endogenous compounds such as steroids. The focus of this project is the identification, characterization, and elucidation of structure-function relationships of the multiple forms of this enzyme. Monoclonal antibodies (MAbs) to rat P-450s are an important tool in these studies. 3-methylcholanthrene (MC)-inducible P0450s have been immunopurified from the livers of rats, C57B1/6 and DBA/2 mice, guinea pigs and hamsters. Their primary structures were compared by amino acid sequence analysis and peptide mapping, and revealed varying degrees of homology. Several previously unidentified amino terminal residues of an immunopurified mouse P-450 were identified, and the sequence of a guinea pig liver P-450 was determined for the first time. A rat lung P-450 was also immunopurified and was indistinguishable from a liver P-450 by several criteria. Using a MAb to ethanol-inducible rat liver P-450, a P-450 has been purified from human liver and was structurally characterized. Since immunopurification typically denatured the P-450s, an alternative approach involving antigen-exchange was developed in which inactive denatured P-450 displaces active P-450 from the immunoadsorbent. Such epitope-specific exchange may be generally applicable to preparation of proteins with retention of activity. The epitopic structure of the P450 surface was mapped by evaluating the binding of MAbs to varius P-450 peptide fragments and by computer-aided alignment of homologous P-450s which are epitopically related. P-450 dependent testosterone metabolism in 3- and 24-month old rats were examined, and hydroxylation patterns as well as content of liver constitutive P-450s varied with age.